Diagnostic Assays for Detecting, Quantifying, and/or Tracking Microbes and Other Analytes

ABSTRACT

The subject invention provides methods and assays for multiplexed detection of analytes using nanocrystals that are uniform in morphology, size, and composition based on their unique optical characteristics. The described methods and assays are particularly useful for detection of microbes and/or microbe-based agents in a complex environmental sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 62/507,895, filed May 18, 2018, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Farming, forestry, and other means of producing food, nutritionaladditives, fiber and natural materials is becoming increasinglydifficult due to numerous environmental challenges. Such challengesinclude pest resistance, extreme temperatures, and pests.

In order to boost yields and protect crops against pathogens, pests, anddisease, farmers have relied heavily on the use of synthetic chemicalsand chemical fertilizers; however, when overused or improperly applied,these substances can run off into surface water, leach into groundwater,and evaporate into the air. As sources of air and water pollution, thesesubstances are increasingly scrutinized, making their responsible use anecological and commercial imperative. Even when properly used, theover-dependence and long-term use of certain chemical fertilizers andpesticides deleteriously alters soil ecosystems, reduces stresstolerance, increases pest resistance, and impedes plant and animalgrowth and vitality.

To empower farmers globally to sustainably grow more food andnutritional supplements as well as foresters to sustainably produce morefiber and structural materials, microorganisms are increasinglyutilized. Microbes such as bacteria, yeast and fungi, and theirbyproducts, are useful in many settings including agriculture, animalhusbandry and forestry, and remediation of soils, water and othernatural resources.

Farmers are increasingly embracing the use of biological agents such aslive microbes, bio-products derived from these microbes, andcombinations thereof, for example, as pesticides. These biologicalagents have important advantages over other conventional pesticides. Theadvantages include: 1) less harmful compared to conventional chemicalpesticides; 2) more efficient and specific; 3) often biodegrade quickly,leading to less environmental pollution.

While enormous potential exists for the use of microbes andmicrobe-based agents, the ability to detect and/or track such microbesand microbe-based agents in the environment has been limited. Theability to detect or trace the microbes and microbe-based agents wouldbe particularly beneficial for agriculture, including for applicationsin growing crops, ornamentals, turf, timber, and animals.

Thus, detection of microbes, including pathogens as well as beneficialmicrobes, or microbe-based agents, in the field would reflect variationsin the environment and promote taking appropriate actions to improveplant health. Moreover, detecting and monitoring microbial pathogens inthe environment can also be beneficial for promoting human health.

Traditional procedures used for detecting microbes typically involveculturing the specimens and detecting microbial activity. In general,the target microbes are inoculated in a culture medium specific to suchtarget microbes, which provides all the nutrients for their growth. Thespecimen may be an untreated natural sample, or it may be a sample thathas been pre-treated by, for example, membrane filtration.

The detection methods commonly utilize at least one analytical reagentthat binds to the specific target and produces a detectable signal.These analytical reagents typically include a probe molecule such as anantibody or oligonucleotide that can bind to the target with a highdegree of specificity and affinity, and a detectable label such as acovalently-linked fluorescent dye molecule that can be detected byproper equipment. Typically, the binding properties of the probemolecule define the specificity of the detection method, and thedetectability of the associated label determines the sensitivity of thedetection method.

Although detection methods with fluorescent dyes possess significantadvantages such as high sensitivity, low background, and accuratemeasurement, and often provide useful results in biomedical research,they are not suitable for detecting and tracking microbes andmicrobe-based agents for the agriculture industry. Reasons include 1)most common fluorophores are aromatic organic molecules that have bothabsorption and emission bands located in the UV/visible portion of thespectrum; 2) the lifetime of the fluorescence emission is usually short,on the order of 1 to 100 ns; 3) it is often not possible to integrate afluorescent signal over a long detection time due to photobleaching; and4) detection of fluorophores requires sophisticated equipment.

Thus, there remains a need for devices and methods to detect and/ortrack beneficial microbes, microbe-based agents, and pathogens in theenvironment quickly and easily, without requiring significant samplepreparation steps, to yield accurate diagnostic information.

SUMMARY OF THE INVENTION

The present invention provides methods and devices to efficiently andaccurately detect, quantify and/or track microbes, microbe-based agents,and/or other analytes in environmental and food samples. The samples maybe, for example, soil, water, oil, waste, food, foliage, and/orbiological samples from livestock or other animals.

The analytes can be microbes, microbe-based agents and/or analytesarising from the presence or activity of microbes. The microbes can bebeneficial microorganisms or pathogens, including agriculturalpathogens.

In preferred embodiments, the present invention provides in-fielddiagnostic assays to quickly, efficiently, and accurately detect,quantify, and/or track analytes of interest. Advantageously, multipleanalytes can be detected simultaneously. Furthermore, the analytes canbe detected at low concentrations, in complex samples, and withnegligible, or no, sample preparation.

Advantageously, the assays of the present invention employ tunablenanocrystals as detection labels to identify the presence, and/orquantify, one or more analytes of interest (e.g., beneficial microbes,microbe-based agents, and/or pathogens). This tunability facilitatesfiltering out background interference, such as from chromophores in asample. This tunability also makes it possible to detect multipleanalytes at the same time. The assay may detect, for example, 1, 2, 3,4, 5, 10, 15, or 20 or more analytes simultaneously from a singlesample.

The nanocrystals are characterized by a uniform morphology and a uniformsize. In addition, the nanocrystals can possess their own unique opticaland magnetic properties such as optical emission spectral profiles,optical absorption spectral profiles, optical power dependency profile,optical lifetime signatures (rise and decay times), and surfacefunctionality. For example, the nanocrystals may be surface modified toenable them to specifically bind to the analyte(s) of interest. Thesurface modification may be achieved by, for example, linking thenanocrystals to antibodies, proteins, aptamers, nucleotides, and/orother compounds.

In one embodiment, the nanocrystals are inorganic luminescent orelectromagnetically active materials that absorb energy acting upon themand subsequently emit the absorbed energy. In one embodiment, thenanocrystals are stokes (down-converting) phosphors. Phosphors thatabsorb energy in the form of a photon and emit a lower frequency (lowerenergy, longer wavelength) band photon are down-converting phosphors.

In another embodiment, the nanocrystals are anti-stokes (up-converting)phosphors. Phosphors that absorb energy in the form of two or morephotons in a low frequency and emit in a higher frequency (higherenergy, shorter wavelength) band are up-converting phosphors.

In one embodiment, the nanocrystals are rare earth (RE)-containingparticles. RE elements include yttrium and the elements of thelanthanide (Ln) series, i.e., lanthanum (La), cerium (Ce), praseodymium(Pr), neodymium (Ne), promethium (Pm), samarium (Sm), europium (Eu),gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium(Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu).

It is advantageous to use nanocrystals with different excitation and/oremission wavelengths, and/or different rise and decay rates, for thedetection of more than one analyte in a single assay.

The method can comprise the steps of: providing an environmental or foodsample suspected of having an analyte of interest, contacting the samplewith a plurality of nanocrystals, and detecting the nanocrystals thatbind to the analyte.

Microbes that can be detected, quantified and/or tracked according tothe subject invention include, but are not limited to bacteria, archaea,yeast, fungi, viruses, protozoa, and multicellular organisms. Themicrobe-based agents that can be analytes according to the subjectioninvention include, but are not limited to, composition containingmicrobes, microbe metabolites and other microbe growth by-products. Inone embodiment, the present invention further provides methods fordetecting a product produced by an entity (such as an animal or plant)in response to a microbe and/or microbe-based agent.

Advantageously, the assays of the subject invention can be utilized tofacilitate tracking of the analytes in the environment or food chain.

The assays of the subject invention can be used in a wide range ofsettings including, but not limited to, crops, livestock, forestry, turfmanagement, ornamentals, pastures, aquaculture, waste treatment, thefood chain, and animal health.

In specific embodiments, the methods of the present invention comprise astep of applying the sample to a substrate to facilitate performing theanalytical assay. The surface of the substrate may have associatedtherewith, for example, antibodies, proteins, aptamers, nucleotides,and/or other compounds that specifically bind to, or otherwise associatewith, the analyte. The assays can utilize, for example, a lateral flowformat, multi-well array, or microfluidics.

In a specific embodiment, the subject invention provides a lateral flowor microfluidic assay format where the nanocrystals in the detectablelabel may be an up-converting phosphor (UCP). In one embodiment, thedetection device detects the up-converting emission wavelength. Inanother embodiment, the detection device detects the phosphor lifetimesignature.

The ability to adjust the size, morphology, absorption, emission, risetime, decay time, power density, and other properties of phosphorparticles, such as up-converting nanocrystals (UCNC) or submicronphosphor particles, enables the formation of materials with a vast arrayof distinctive signatures. The versatility of the rare earth UCNCplatform significantly increases the ability to have a broad detectioncapability using a single reader system. Additionally, the ability tooptically tune the rare earth nanoparticle or submicron particle uniquespectral fingerprints provides highly advantageous multiplexingcapabilities.

The methods of the subject invention facilitate rapid, sensitive, andinexpensive, detection and/or quantification of microbes and/ormicrobe-based agents of interest in complex samples. The use ofnanocrystals as labels according to the subject invention provides arapid, multiplexed and specific assay platform capable of detecting lowlevels of analyte targets in complex environmental and food samples,such as, for example, in the case of food, agriculture, and livestocksamples.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and devices to efficiently andaccurately detect, quantify, and/or track microbes, microbe-basedagents, and/or other analytes. The analytes can be detected inenvironmental or food samples, such as in soil, water, food, waste, oil,plants and biological samples from animals. The microbes can be, forexample, beneficial microorganisms or pathogens, including agriculturalpathogens and animal pathogens.

The methods of the present invention employ nanocrystals as detectionlabels to detect one or more analytes of interest (e.g., beneficialmicrobes, microbe-based agents, and/or pathogens). Advantageously,multiple analytes can be detected simultaneously in a single assay.

According to the present invention, the nanocrystals exhibit tunablephysical properties and, advantageously, have controlled sizeuniformity, shape selectivity and surface functionality. For example,the nanocrystals may be surface modified to enable them to specificallybind to an analyte of interest. The surface modification may be achievedby linking the nanocrystals to, for example, antibodies, proteins,aptamers, nucleotides, and/or other compounds.

In one embodiment, the present invention provides methods for detectingan analyte in a sample comprising the steps of:

contacting the sample with a plurality of nanocrystals, wherein thenanocrystals have been surface modified with an entity that specificallybinds to the target analyte,

separating the nanocrystals bound to the analyte from unboundnanocrystals, and

detecting the nanocrystals that bind to the analyte.

The microbes detected, quantified and/or tracked according to thesubject invention can be any prokaryotic or eukaryotic microscopicorganism, including, but not limited to bacteria (e.g., spore orvegetative, Gram positive or Gram negative), archaea, yeast, fungi(e.g., filamentous fungi and fungal spores), viruses, protozoa, ormulticellular organisms. In some cases, the microorganisms of particularinterest are those that are pathogenic. The term “pathogen” is used torefer to any pathogenic microorganism. In other instances the microbe isbeneficial.

In a specific embodiment, the method is used to detect, optionally in acomplex environmental sample, pathogens that cause citrus greeningdisease. Citrus greening disease also known as Huanglongbing (HLB) iscaused by the phloem-limited fastidious prokaryotic α-proteobacteriumCandidatus Liberibacter spp., Ca. africanus, and Ca. L. americanus.

The methods described herein are suitable for use on any tree or otherplant that is infected or may be infected with citrus greening disease.Exemplary plants include, but are not limited to, any cultivar from thegenus Citrus, including but not limited to Citrus sinensis (naveloranges), lemon (C. limon), lime (C. latifolia) grapefruit (C.paradise), sour orange (C. aurantium), and mandarin (C. reticulata).

In other specific embodiments, the assays of the subject invention areused to detect, quantify and/or track the plant pathogens that causePotato Late Blight, Grape Powdery Mildew, Red Blotch, Tobacco MosaicVirus, Fire blight and/or Pierce's Disease.

The sample can be, but is not limited to, water, soil, food, plant, air,waste, biological samples from animals, dust, and samples collected fromsurfaces.

Collection may be achieved by any of a variety of methods, including,but not limited to, use of a sponge, wipe, swab (e.g., a wound fiberproduct), film, brush (e.g., having rigid or deformable bristles), andthe like, and combinations thereof.

In one embodiment, the analyte is a microbe-based agent. Microbe-basedagents according to the subjection invention include, but are notlimited to, composition that contain microbes, microbe metabolites andother microbe growth by-products. In specific embodiments, themicrobe-based agent is a microbial biosurfactant or mycotoxin.

The assays of the subject invention can be utilized to facilitatetracking of microbes, microbe-based agents, and other analytes in theenvironment or food chain.

In preferred embodiments, the nanocrystals are monodisperse particles incrystalline form having a rare earth-containing lattice, uniformthree-dimensional size, and uniform polyhedral morphology. Preferably,the monodisperse particles are capable of self-assembly intosuperlattices due to their uniform size and shape.

In one embodiment, the nanocrystals are inorganic luminescent orelectromagnetically active materials that absorb energy acting upon themand subsequently emit the absorbed energy. Such nanocrystals can act asphosphors that continue to emit light for greater than 10⁻⁸ secondsafter the removal of the absorbed light. The half-life of the afterglow,or phosphorescence, of a phosphor typically ranges from about 10⁻⁶seconds to days.

In certain embodiments, the nanocrystals according to the subjectinvention are stokes (down-converting) phosphors. Phosphors that absorbenergy in the form of a photon and emit a lower frequency (lower energy,longer wavelength) band photon are down-converting phosphors.

In other embodiments, the nanocrystals are anti-stokes (up-converting)phosphors. Phosphors that absorb energy in the form of two or morephotons in a low frequency and emit in a higher frequency (higherenergy, shorter wavelength) band are up-converting phosphors.Up-converting phosphors can be, for example, irradiated by nearinfra-red light, a lower energy, longer wavelength light, and emitvisible light that is of higher energy and a shorter wavelength.

In one embodiment, the nanocrystals are rare earth (RE)-containingparticles. RE elements include yttrium and the elements of thelanthanide (Ln) series, i.e., lanthanum (La), cerium (Ce), praseodymium(Pr), neodymium (Ne), promethium (Pm), samarium (Sm), europium (Eu),gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium(Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu).

In certain embodiments, the down-converting nanocrystals of theinvention can be excited at a wavelength between 1 nm and 400 nm,preferably, between 10 nm and 400 nm.

In other embodiments, the up-converting nanocrystals of the inventioncan be excited at a wavelength between 700 nm and 2000 nm, preferably,between 800 nm and 1500 nm, more preferably, 900 nm and 1000 nm. In aspecific embodiment, the up-converting nanocrystals can be excited at awavelength from 960 nm to 980 nm.

In one embodiment, the nanocrystals of the invention emit light at awavelength from 400 nm to 12,000 nm.

In one embodiment, the nanocrystals used in the present assay may becombined with a second reporter such as quantum dots, carbon nanotubes,as well as magnetic and dye-doped nanoparticles. Combining nanocrystalswith other waveshifting and absorbing materials allows for additionalmultiplexing and functionality. Two complimentary particles such as anupconverting nanocrystal and a downconverting quantum dot that absorbsthe emission of the upconverting nanocrystal with the same captureantibodies will bind to a target. When activated with a 980 nm light thequantum dot by itself does not emit but when in proximity of aupconverting nanocrystal, the nanocrystal will transfer the necessaryenergy to activate the quantum dot. The only time the two particles areclose enough is if they bind to a specific target. In a microfluidicsystem, binding effects can be quantified in real time.

Adding magnetic properties to the nanocrystals allows for fasterprocessing time before analysis as the particles can be funneled intothe assay with a magnet. The magnetic properties can also be read duringdetection. Rare-Earth crystals combined with other metals exibitdifferent properties such as paramagnetic and ferromagnetic.

Organic dyes coated over the nanocrystals form a filter and can benefitspectral interference. Lanthanide lines sometimes overlap and addingorganic materials allows for blocking of certain regions in the spectrumto produce single emissions.

Multiple nanocrystals possessing distinct sizes, lifetimes and/ormorphologies can be combined and introduced into or onto a complexenvironmental sample providing multiple unique detectable labels thatcan be used for multiple analyte detections. The rare earth nanocrystalsare advantagous because of their relatively long phosphorescencelifetime decays attributed to, for example, the trivalent rare earth (orlanthanide) metals.

It is advantageous to use nanocrystals with different excitation and/oremission wavelengths for the detection of more than one analyte in asingle assay by using different labels to identify particular targets.For example, it is possible to generate multiple spectrally-separatecolors (e.g., blue, green, and red) by means of infrared (IR), ultraviolet (UV), or electron excitation to measure phosphor emissionwavelengths, intensity amplitudes, and the number of analytes at thesame time. In particular, the immunocytochemical use of nanocrystalconjugates with capture molecules allows a sensitive detection of smallquantities of analyte in the environmental samples.

Advantageously, the multiplexing property of the assay usingnanocrystals makes it possible to detect an analyte of interest in acomplex environmental or food sample without interference from samplecomponents. For example, nanocrystals with tunable characteristic allowthe quantification of analytes of interest from interfering chromophoresthat are present in soil or plant samples.

In one embodiment, the subject invention also provides a method for thepreparation of the nanocrystals. The method employs the steps of: in areaction vessel, dissolving at least one precursor metal salt in asolvent to form a solution; placing the reaction vessel in a heated saltbath having a temperature of at least about 340° C.; applying heat tothe salt bath to rapidly decompose the precursor metal salts in thesolution to form the monodisperse particles; keeping the reaction vesselin the salt bath for a time sufficient to increase the size of themonodisperse particles; removing the reaction vessel from the salt bath;and quenching the reaction with ambient temperature solvent.

Advantageously, the present invention provides a sensitive assay with adetection sensitivity for microbe at 10³ CFU/mL and lower. In preferredembodiments the sensitivity is 10² CFU/mL, more preferably, 10¹ CFU/mL.Thus, the assay can detect microbes in a complex sample ranging from 10¹CFU/mL to 10⁹ CFU/mL and higher.

The present invention also provides a sensitive assay with a detectionsensitivity for microbe-based agents as low as 0.001 ng/mL.

Advantageously, the assays can be performed in the field. In certainembodiments, the assays are performed within 1000, 500, 250, 100, 50,20, 10, 5 or even 1 yard or less from wherein the sample was obtained.Further, the assay may be performed, for example, within 60, 45, 30, 20,10, 5, or even 1 minute or less from when the sample was taken.

In one embodiment, the methods can be used for simultaneously detectingone or more analytes in a complex environmental sample. The detectioncan be accomplished in 60 minutes or less, 50 minutes or less, 40minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes orless, or 5 minutes or less. In preferred embodiments, the assay isconducted more quickly and/or with less sample preparation than assaysutilizing PCR or standard ELISA. The results may be read immediatelyupon completion of the assay and/or stored and/or transmitted to anotherlocation. For example, the results may be transmitted electronically forstorage and/or further analysis. The results may be, for example,transmitted to an electronic storage cloud or other stored database.

These tools can be used to conduct quality control and assess productspecifications both immediately following production as well as at afarmer's field just prior to application. This facilitates rapid productrelease that is highly beneficial in a local microbial fermentationsystem, as well as in any system, because it is faster, cheaper, andmore accurate than other current methods.

The assays of the subject invention can also be used to confirm thecharacteristics of a microbial product purchased by a consumer. Thisaspect of the invention has great value as many biologicals lose potencyover time and become well below stated potency by the time they arebought or used. This aspect also helps to manage inventory, anddetermine which products are off specification for products with singlemicrobes or those that contain several.

A plant's nutrition, growth, and proper functioning are dependent on thequantity and distribution of robust populations of natural microflorathat, in turn, are influenced by soil fertility, tillage, moisture,temperature, aeration, organic matter, and many other factors. Prolongeddrought, variable rainfall, and other environmental variations,including the proliferation of nematodes and other pests, influencethose factors and affect soil diversity and plant health. Theseenvironmental variables manifest themselves in multiple dimensions,including geography, seasonality in a given year, and differencesbetween years. They also exist within a specific farm and even within assmall an area as an acre, or less or between animal species or evenindividual animals within a species. Using the assays of the subjectinvention to analyze, quickly and accurately, microbial (beneficial andpathogenic) presence and ecology within meta and micro environmentsprovides much greater power to farmers, regulatory officials, complianceofficials, basic producers, distribution agents in the supply chain andother organizations or individuals wishing to better enhance theirassets, manage pathogens, and optimize the efficiency and economicperformance of their business.

Nanocrystals

The nanocrystals, useful according to the subject invention, areinorganic luminescent or electromagnetically active materials thatabsorb energy acting upon them and subsequently emit the absorbedenergy. Such nanocrystals can act as phosphors that continue to emitlight for greater than 10⁻⁸ seconds after the removal of the absorbedlight. The half-life of the afterglow, or phosphorescence, of a phosphortypically ranges from about 10⁻⁶ seconds to days.

The nanocrystals of the invention may have different optical propertiesbased on their composition, their size, and/or their morphology (orshape). In one embodiment, the invention relates to a combination of atleast two types of nanocrystals, where each type is a plurality ofmonodisperse particles having a single pure crystalline phase of a rareearth-containing lattice, a uniform three-dimensional size, and auniform polyhedral morphology; and where the types of monodisperseparticles differ from one another by composition, by size, or bymorphology. In a preferred embodiment, the types of monodisperseparticles have the same composition but different morphologies.

In one embodiment, the nanocrystals according to the subject inventionare stokes (down-converting) phosphors. Phosphors that absorb energy inthe form of a photon and emit a lower frequency (lower energy, longerwavelength) band photon are down-converting phosphors.

In another embodiment, the nanocrystals are anti-stokes (up-converting)phosphors. Phosphors that absorb energy in the form of two or morephotons in a low frequency and emit in a higher frequency (higherenergy, shorter wavelength) band are up-converting phosphors.Up-converting phosphors, for example, are irradiated by near infra-redlight, a lower energy, longer wavelength light, and emit visible lightwhich is of higher energy and a shorter wavelength.

In one embodiment, the nanocrystals are rare earth (RE)-containingparticles. RE elements include yttrium and the elements of thelanthanide (Ln) series, i.e., lanthanum (La), cerium (Ce), praseodymium(Pr), neodymium (Ne), promethium (Pm), samarium (Sm), europium (Eu),gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium(Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu).

In a specific embodiment, the nanocrystals of the invention have a rareearth-containing lattice that may be an yttrium-containing lattice or alanthanide-containing lattice. The lattice contains yttrium (Y) or alanthanide (Ln) in its +3 oxidation state. The charge is balanced in thelattice by the presence of an anion such as a halide (fluoride, F, beingpreferred), an oxide, an oxysulfide, an oxyhalide (e.g., OCl), asulfide, etc. Alkali metals, i.e., lithium (Li), sodium (Na), potassium(K), rubidium (Rb), and cesium (Cs) and/or alkali earth metals beryllium(Be), magnesium (Mg) calcium (Ca), strontium (Sr), and barium (Ba) mayalso be a component of the host lattice. The alkali metals or alkalineearth metals are often called “lattice modifiers.”

The nanocrystals may vary in size. In one embodiment, crystals of theinvention may be described as nanocrystals with their largest dimensionranging approximately 1 nm to 1,000 nm in size, preferably, from 5 nm to750 nm, more preferably, from 10 nm to 500 nm, most preferably, 20 nm to400 nm. Large crystals, with at least one dimension of approximately 1μm to 400 μm, represent another embodiment of the invention. The size ofthe crystal depends on the stoichiometric ratio of elements making thecrystal or the stoichiometric ratio precursor used to prepare theparticle as well as the length of reaction time.

Nanocrystals used according to the subject invention preferably have asingle pure crystalline phase of a RE-containing lattice. In oneembodiment, the nanocrystal is a α, β, or cubic-phase crystal. In apreferred embodiment, the nanocrystal is a hexagonal (β)-phase particle.

For the synthesis of monodisperse particles of the invention, the alkalimetal or alkaline earth metal present in the lattice may determine thecrystal symmetry providing morphological control over the particles aswell as independent tunability of a particle's other properties, such asthe optical properties of a luminescent particle. For example, thecrystal symmetry of LiYF₄, NaYF₄, and KYF₄ are tetragonal, hexagonal,and trigonal, respectively.

The chemical composition of the particles of the invention providesunique polyhedral morphologies. Representative yttrium-containinglattices include, but are not limited to LiYF₄, BaYF₅, BaY₂F₈NaYF₄,KYF₄, Y₂O₂S, Y₂O₃, and the like. The lanthanide-containing lattice maybe one having any element of the lanthanide series. Representativelanthanide-containing lattices include, but are not limited to, LaF₃,CeF₃, PrF₃, NeF₃, PmF₃, SmF₃, EuF₃, GdF₃, TbF₃, DyF₃, HoF₃, ErF₃, TmF₃,YbF₃LuF₃, NaGdF₄, Gd₂OS₃, LiHoF₄, LiErF₄, CeO, SrS, CaS, GdOCl, and thelike.

In one embodiment, the chemical composition of the particles may containdopants and lattice modifiers, which impart unique properties to thecomposition.

The morphology of the nanocrystals can be spherical, hexagonal, cubic,rod-shaped, diamond-shaped, odd shape such as a mushroom or a dumbbell.Advantageously, UCNC do not photobleach and allow high power densityexcitation over long term exposure with simultaneous signal integration.They can be stored indefinitely without a decrease in light emittingefficiency and thus they allow repeated irradiation and analysis. Unlikeprevious inorganic markers of the past, the nanocrystals are uniform andprovide a consistent signal based upon their concentration. If thecrystals are amorphous the distribution of the atoms is not consistent,there are defects in the structures and the emitted optical signalcannot be quantified. The invention takes advantage of the uniformmorphology of the crystals. Similar to a remote control, an infraredpulsed light is emitted from the crystal during the test. Suchproperties facilitate the quantification of analytes of interest in acomplex environmental sample.

A. Down-Converting Phosphors

Down-converting phosphor materials include RE element doped oxides, REelement doped oxysulfides, RE element doped fluorides. Examples ofdown-converting phosphors include, but are not limited to Y₂O₃:Gd,Y₂O₃:Dy, Y₂O₃:Tb, Y₂O₃:Ho, Y₂O₃:Er, Y₂O₃:Tm, Gd₂O₃:Eu, Y₂O₂S:Pr,Y₂O₂S:Sm, Y₂O₂S:Eu, Y₂O₂S:Tb, Y₂O₂S:Ho, Y₂O₂S:Er, Y₂O₂S:Dy, Y₂O₂S:Tm,Y₂O₂S:Eu (red), Y₂O₃:Eu (red), and YV0 ₄:Eu (red). Other examples ofdown-converting phosphors are sodium gadolinium fluorides doped withother lanthanides, e.g., NaGdF₄:Tb, wherein the Tb can be replaced withEu, Dy, Pr, Ce, etc. Lanthanide fluorides are also known asdown-converting fluorides, e.g., TbF₃, EuF₃, PrF₃, and DyF₃.

B. Up-Converting Phosphors

Up-converting phosphors derived from RE-containing host lattices, suchas described above, doped with at least one activator couple comprisinga sensitizer (also known as an absorber) and an emitter. Suitableup-converting phosphor host lattices include: sodium yttrium fluoride(NaYF₄), lanthanum fluoride (LaF₃), lanthanum oxysulfide, REoxysulfide(RE₂O₂S), RE oxyfluoride (RE₄O₃F₆), RE oxychloride (REOCl),yttrium fluoride (YF₃), yttrium gallate, gadolinium fluoride (GdF₃),barium yttrium fluoride (BaYF₅, BaY₂F₈), and gadolinium oxysulfide,wherein the RE can be Y, Gd, La, or other lanthanide elements. Suitableactivator couples are selected from: ytterbium/erbium,ytterbium/thulium, and ytterbium/holmium. Other activator couplessuitable for up-conversion may also be used.

By combination of RE-containing host lattices with just these threeactivator couples, at least three phosphors with at least threedifferent emission spectra (red, green, and blue visible light) areprovided. Generally, the absorber is ytterbium and the emitting centercan be selected from: erbium, holmium, terbium, and thulium; however,other up-converting phosphor particles of the invention may containother absorbers and/or emitters. The molar ratio of absorber:emittingcenter is typically at least about 1:1, more usually at least about 3:1to 5:1, preferably at least about 8:1 to 10:1, more preferably at leastabout 11:1 to 20:1, and typically less than about 250:1, usually lessthan about 100:1, and more usually less than about 50:1 to 25:1,although various ratios may be selected by the practitioner on the basisof desired characteristics (e.g., chemical properties, manufacturingefficiency, excitation and emission wavelengths, quantum efficiency, orother considerations). For example, increasing the Yb concentrationslightly alters the absorption properties, which is useful forbiomedical applications. Additionally, the introduction of other rareearth and transition metal dopants, alterations in the dopingconcentrations, and host lattice modifications, all provide furthertunability over spectral profiles as well as rise and decay times.

The optimum ratio of absorber (e.g., ytterbium) to the emitting center(e.g., erbium, thulium, or holmium) varies, depending upon the specificabsorber/emitter couple and desired spectral profile and lifetime. Forexample, the absorber:emitter ratio for Yb:Er couples is typically inthe range of about 1:1 to about 100:1, whereas the absorber:emitterratio for Yb:Tm and Yb:Ho couples is typically in the range of about500:1 to about 2000:1. These different ratios are attributable to thedifferent matching energy levels of the Er, Tm, or Ho with respect tothe Yb level in the crystal. For most applications, up-convertingphosphors may conveniently comprise about 10-30% Yb and either: about1-2% Er, about 0.1-0.05% Ho, or about 0.1-0.05% Tm for optimal quantumefficiency, although other formulations may be employed.

In some embodiments, inorganic phosphors are optimally excited byinfrared radiation of about 900 to 1000 nm, preferably about 960 to 980nm. For example, but not by limitation, a microcrystalline inorganicphosphor of the formula YF₃:Yb_(0.10)Er_(0.01)exhibits a luminescenceintensity maximum at an excitation wavelength of about 980 nm.Up-converting phosphors of the invention typically have emission maximathat are in the visible to near infrared range. For example, specificactivator couples have characteristic emission spectra: ytterbium-erbiumcouples have emission maxima in the red (660 nm) or green (540 nm)portions of the visible spectrum, depending upon the phosphor host;ytterbium-holmium (535 nm) couples generally emit maximally in the greenportion, ytterbium-thulium typically have an emission maximum in theblue (480 nm), red (635 nm) and infrared (800 nm) range, andytterbium-terbium usually emit maximally in the green (545 nm) range.For example, Y_(0.80) Yb_(0.19) Er_(0.01)F, emits maximally in the greenportion of the spectrum.

The phosphor particle of the invention can be excited at 915 nm insteadof 980 nm where the water absorption is much higher and more tissueheating occurs. The ratio(s) chosen will generally also depend upon theparticular absorber-emitter couple(s) selected, and can be calculatedfrom reference values in accordance with the desired characteristics. Itis also possible to control particle morphologies by changing the ratioof the activators without the emission properties changing drasticallyfor most of the ratios but quenching may occur at some point.

C. Particle Properties Based on Composition, Morphology, and Size

Properties of the monodisperse particles can be tuned in a variety ofways. The properties of the monodisperse particles, the characteristicabsorption and emission spectra, may be tuned by adjusting theircomposition, e.g., by selecting a host lattice, and/or by doping.Advantageously, given their uniform polyhedral morphology, themonodisperse particles exhibit anisotropic properties. Particles of thesame composition but different shape exhibit different opticalproperties due to their shape and/or size.

In one embodiment, the monodisperse particles are varied in compositionand/or shape to give different decay lifetimes. Having differentspectral decay lifetimes allows unique phosphor particles to bedifferentiated from one another. The ability to have monodisperseparticles of the same composition but different morphologies accordingto the invention permits use of one composition (especially in regulatedindustries such as pharmaceuticals or medical devices) but todistinguish its morphologies through their unique optical properties.

Thus, in addition to the characteristic absorption and emission spectrathat can be obtained the rise and decay times of a monodisperse particleof the invention can also be tuned by particle size and morphology. Therise time is measured from the moment the first excitation photon isabsorbed to when the first emission photon is observed. The decay timeis measured by the slope of the emission decay, or the time it takes forthe phosphor to stop emitting once the excitation source is turned off.This is also described as the time it takes for depletion of electronsfrom the excited energy levels. By changing the dopant ratio, the riseand decay times can be reliably altered.

Typically, an excited state population decays exponentially afterturning off the excitation pulse by first-order kinetics, following thedecay law, I(t)=I₀ exp (−t/τ), whereby for a single exponential decayI(t)=time dependent intensity, I₀=the intensity at time 0 (oramplitude), and τ=the average time a phosphor (or fluorophor) remains inthe excited state (or <t>) and is equal to the lifetime. (The lifetime τis the inverse of the total decay rate, τ(T+k_(nr))⁻¹, where at time tfollowing excitation, T is the emissive rate and k_(nr) is thenon-radiative decay rate). In general, the inverse of the lifetime isthe sum of the rates which depopulate the excited state. Theluminescence lifetime can be simply determined from the slope of a plotof lnl(t) versus t (equal to l/τ). It can also be the time needed forthe intensity to decrease to lie of its original value (time 0). Thus,for any given known emission wavelength, a number of parameters fittingthe exponential decay law can be monitored to identify a particularphosphor or group of phosphors, thus permitting their use, for example,in developing unique anti-counterfeiting codes, signatures, orlabels/taggants.

In most instances, lifetimes are controlled by variations in the crystalcomposition or overall particle size. However, by controlling theparticle morphology and uniformity as with the monodisperse particles ofthe invention one can create particles of visually distinct morphologiespossessing lifetimes that are unique to that morphology whilemaintaining identical chemical compositions among the variousmorphologies. This feature allows for a highly complex optical signatureor taggant which, may be used in serialization and multiplexing assaysor analysis in various fields such as, for example, assays, biomedical,optical computing, as well as use in security and authentication.

Particle size and morphology may be controlled by varying reactionconditions such as stoichiometric precursor metal salt ratio, heatingrate of the salt bath, and reaction time. The initial rate of heating inthe salt bath is important in determining the morphology by selectingwhich crystal planes will undergo the most rapid growth. Final particlesize is determined by total reaction time in the salt bath as well asprecursor ratios. After the reaction vessel reaches the temperature ofthe salt bath, the longer the time the vessel remains in the salt baththe larger the particles may grow.

D. Superlattice Assembly

Due to their uniformity in size and morphology, the monodisperseparticles of the invention are able to self-assemble into superlatticestructures. These superlattice structures represent the lowest freeenergy conformation for the assemblage. This uniform build-up isaccomplished with monodisperse particles of uniform size and morphologyas according to the invention. The superlattices form via interfacialself-assembly, building hierarchical structures with orders on differentlength scales.

Superlattices of the monodisperse particles of the invention may beformed by suspending the particles in a solvent and then drop-castingthem onto a surface. As the solvent slowly evaporates, the particlesarrange themselves into a superlattice with both positional andorientational order. Any solvent which disperses the particles may beused, such as, but not limited to, benzene, carbon tetrachloride,chlorobenzene, chloroform, cyclohexane, dimethyl-formamide, dimethylsulfoxide, ethanol, heptane, hexane, pentane, tetrahydrofuran, toluene,with nonpolar organic solvents such as hexane being preferred.

Superlattices of the invention may be transparent films of themonodisperse particles of the invention, particularly with monodispersenanoparticles of the invention. In order to form a superlattice theconstituent particles must be of identical or nearly identical size andshape. When both conditions are met a uniform, patterned, monolayer ofparticles forms. Advantageously, the monodisperse particles of theinvention meet these criteria for uniform size and uniform morphology.Due to the small size and uniformity of the particles of the invention,there is no scattering of light and as a result a transparent film isobtained.

Functionalization of the Nanocrystals

In one embodiment, the nanocrystals have been functionalized with one ormore capture molecules. This can be done by, for example, linking thenanocrystals to antibodies, proteins, polypeptides, aptamers,nucleotides, and/or other compounds that specifically bind to an analytesuch as a target microbe or a microbe-based agent. In anotherembodiment, the analyte target could also be any of a range of hostbiomolecules induced to express in response to infection by a pathogenicmicroorganism.

“Specific,” as used herein, refers to an antibody, or other entity, thatonly recognizes the target to which it is specific or that hassignificantly higher binding affinity to the target to which it isspecific compared to binding to molecules to which it is non-specific.The binding affinity measures the strength of the interaction between anepitope and an antibodies antigen binding site. Higher affinityantibodies will bind a greater amount of antigen in a shorter period oftime than low-affinity antibodies. Thus, the binding affinity constantcan vary widely from below 10⁵ mol⁻¹ to above 10¹² mol⁻¹.

In a preferred embodiment, the antibody may comprise a complete antibodymolecule having full length heavy and light chains or a fragment thereofand may be, but are not limited to, Fab, modified Fab, Fab′, modifiedFab′, F(ab′)2, Fv, single domain antibodies (e.g., VH or VL or VHH),scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies,triabodies, tetrabodies and epitope-binding fragments of any of theabove (see, for example, Holliger and Hudson, 2005, Nature Biotech.23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews—Online2(3), 209-217). The antibodies can be specific to, for example,proteins, or epitopes of proteins, that is expressed at the surface ofthe target microbes.

Antibodies, including chimeric antibodies, can be used according to thesubject invention. Chimeric antibodies are those antibodies encoded byimmunoglobulin genes that have been genetically engineered so that thelight and heavy chain genes are composed of immunoglobulin gene segmentsbelonging to different species. These chimeric antibodies can be lessantigenic. Multi-valent antibodies may comprise multiple specificitiesor may be monospecific.

The antibodies for use in the present invention can be purchased or theycan be generated using various methods, including phage display methods,known in the art. Also, mice, or other organisms, including othermammals, may be used to express antibodies.

The antibody can be of any class (e.g., IgG, IgE, IgM, IgD and IgA) orsubclass of immunoglobulin molecule. In one embodiment the antibody foruse in the present invention is of the IgG class and may be selectedfrom any of the IgG subclasses IgG1, IgG2, IgG3 or IgG4.

The antibody for use in the present invention may include one or moremutations to alter the activity of the antibody.

Examples of antigens include, but are not limited to, cell surfacemolecules that are stable or transient plasma membrane components,including peripheral, extrinsic, secretory, integral or transmembranemolecules. In some embodiments, the molecule is exposed at the exteriorof the plasma membrane of the cell. In other embodiments, the antigenicdeterminant is not surface exposed but is instead exposed upon, forexample, cell lysis. In certain embodiments, the antigen is a moleculeof known structure and having a known or described function, includingbut not limited to glycoproteins, lipoproteins, and cell wall anchoredproteins; the epitope of the antigen may also be a non-protein basedbiomolecule

In another embodiment, the surface antigen and/or the epitope of thesurface antigen may be selected based on genome sequence information.The identification of antigens may involve biological software known inthe art (see, for example, Bioinformatics Approach for Cell SurfaceAntigen Search of Helicobacter pylori, Ragini Tiwari et al., Journal ofPharmacy Research 2012, 5(11), 5184-5187) based on the sequenceinformation of specific motif of interest. For example, programs likeSignalP, LipoP, PSORTb, and TMHMMS can be used to filter and select anantigen of interest.

Specifically, SignalP 4.1 server predicts the presence and location ofsignal peptide cleavage sites in amino acid sequences from differentorganisms: Gram-positive prokaryotes, Gram-negative prokaryotes, andeukaryotes. The method incorporates a prediction of cleavage sites and asignal peptide/non-signal peptide prediction based on a combination ofseveral artificial neural networks. The website address is:www.cbs.dtu.dk/services/SignalP.

The TMHMM server predicted membrane spanning helices in proteins bysearching hydrophobic amino acids. The algorithm predicted number ofhelices and highlighted spanning the length of the peptides. The webaddress is: www.cbs.dtu.dk/services/TMHMM. The lipoP server predictedlipoproteins by available lipoprotein signal peptides. The web addressis: www.cbs.dtu.dk/services/LipoP.

PSORTb predicts the localization site and the associated probability.Subcellular localization of proteins has been done based on amino acidsequence information. A protein subcellular localization was influencedby several features present within the protein's primary structure, suchas the presence of a signal peptide or membrane-spanning alpha-helices.The web address is: http://www.psort.org/psortb/.

Advantageously, the methods of the subject invention can be used todetect microbes that are difficult or impossible to grow in culture, orthat can be grown in culture but only very slowly. Because the methodsof the subject invention can detect very low numbers of microbes, it isnot necessary to grow the microbes from a sample in a culture toincrease their numbers prior to performing the assay of the subjectinvention. Accordingly, the assay of the subject invention can be usedto detect microbes that not amendable for cultivarion under standardlaboratory conditions, or in culture take longer than 1, 2, 5, 10, 24,72 or more hours to double in number, or which cannot be grown at all inculture. Thus, the assay of the subject invention can be used to detect,quantify and/or track beneficial microbes such as pasteuria, as well asthe pathogens that cause citrus greening disease and zebra chip disease.Viruses can also be detected.

The capture molecules for such difficult-to-culture microbes can bebased on antigens identified as described above, as well as throughmetagenome sequencing. Metagenomics is the study of genetic materialrecovered directly from environmental samples. Conventional sequencingrequires a culture of identical cells as a source of DNA. However, manymicroorganisms in environmental samples cannot be cultured and thuscannot be sequenced. Advances in bioinformatics, refinements of DNAamplification, and increases in computational power have greatly aidedthe analysis of DNA sequences recovered from environmental samples,allowing the adaptation of shotgun sequencing to metagenomic samples.The random nature of shotgun sequencing ensures that many of theseorganisms, which would otherwise go unnoticed using traditionalculturing techniques, will be represented by at least some sequencesegments.

The genomes of pathogenic microorganisms often contain pathogenicityislands acquired through horizontal gene transfer. These gene islandsare incorporated into the genome of pathogenic organisms, but aretypically absent from non-pathogenic related species. Pathogenicityisland DNA sequences often code for virulence factors which areexcellent targets for specific antibodies. These pathogenicity islandsequences can be identified via bioinformatic analysis, subcloned,expressed and used as pure antigen for generating antibodies.

A first step of metagenomic data analysis often entails the execution ofcertain pre-filtering steps, including the removal of redundant,low-quality sequences and sequences of probable eukaryotic origin. Next,metagenomic analysis typically use two approaches in the annotation ofcoding regions in the assembled contigs. The first approach is toidentify genes based upon homology with genes that are already publiclyavailable in sequence databases, by simple BLAST searches. The second,ab initio, uses intrinsic features of the sequence to predict codingregions based upon gene training sets from related organisms. This isthe approach taken by programs such as GeneMark and GLIMMER. Thisapproach facilitates the detection of coding regions that lack homologsin the sequence databases.

Metagenomic sequencing is particularly useful in the study of viralcommunities. As viruses lack a shared universal phylogenetic marker (as16S RNA for bacteria and archaea, and 18S RNA for eukarya), the only wayto access the genetic diversity of the viral community from anenvironmental sample is through metagenomics.

In accordance with the subject invention, metagenome sequencing can beperformed, for example, on a leaf sample having a complex mixture ofmicrobes. Metagenome sequencing can be used to identify DNA codingsequences that can then be cloned and engineered to express peptidesand/or full proteins that can then be used to generate antibodies foruse in lateral flow assays (or other assays) for detecting, quantifyingand/or tracking an uncultureable microbe.

In one embodiment, the surface of nanocrystals may be coated with asurface modifier, for example, polymers such as polyacrylic acid andcopolymers such as maleic acid/polyacrylic acid and block copolymers, oran inert silica layer to allow or improve the conjugation of the capturemolecule to the particle surface. The nanocrystals conjugated to eachtype of capture molecule have unique and uniform morphology, size,and/or composition, producing a unique optical lifetime signature.

In one embodiment, the conjugation of the capture molecule is achievedusing a method known in the art. Generally, conjugation is accomplishedusing a carboxylic acid activating reagent for coupling to nuclephiles.In a specific embodiment, the conjugation of the capture molecules isachieved via the N-hydroxysuccinimide (NHS) and/or Sulfo-NHS forpreparing amine-reactive esters of carboxylate groups for chemicallabeling, crosslinking and solid-phase immobilization. In additional toNHS esters and thiols, imidoesters can also be used as amine-specificfunctional groups that are incorporated into reagents for proteincrosslinking and labeling.

Assay Formats

In specific embodiments, the methods of the present invention comprise astep whereby target microbes and/or microbe-based agents in anenvironmental sample become affixed to, or otherwise associated with, asubstrate. This step can be accomplished by, for example, treating thesurface of a substrate with capture molecules, for example, antibodies,proteins, nucleotides, and other compounds that specifically recognizethe target microbe and/or the microbe-based agent. The capture moleculesmay be the same or different molecules used for functionalizing thesurface of nanocrystals to specifically capture the target of interest.

A separating step according to the subject invention may be achievedthrough methods known in the art. The separation method may involve, butis not limited to, wash, perfusion, and dialysis.

Although not generally necessary, in certain embodiments of the subjectinvention enrichment techniques such as the use of paramagnetic UCNCscan be used to enrich the sample, thereby further enhancing sensitivityand/or selectivity.

A. Lateral Flow Assays

In a preferred embodiment, the present invention employs a lateral flowassay, which is utilized to test for the presence, absence, and/orquantity of an analyte of interest in a sample. In one embodiment a“sandwich” assay is used whereby an antibody (or other binding liquid)is immobilized on a solid support to capture a target analyte therebyfacilitating the detection and/or quantification by observing boundanalyte.

In one embodiment, the assay of the invention is performed on a lateralflow test strip. Lateral flow test strips have a solid support on whichthe sample-receiving area and the target capture zone(s) are located.The solid support also provides for capillary flow of sample out fromthe sample receiving area to the target capture zone(s) when the lateralflow test strip is exposed to an appropriate carrier liquid of thesample. The materials of such solid support can be, for example, organicor inorganic polymers, and natural and synthetic polymers. More specificexamples of suitable solid supports include, but are not limited to,glass fiber, cellulose, nylon, crosslinked dextran, variouschromatographic papers, Diomat™ and nitrocellulose. In a preferredembodiment, the material of the solid support is nitrocellulose. In afurther embodiment, the lateral flow test strips may contain one or moretarget capture zones.

In one embodiment, the lateral flow test strips are constructed for usewith a device that directs a particular wavelength of light, forexample, infrared, visible, UV light, or with an electron beam, and inturn captures the return wavelength emitted by the nanocrystals whenstimulated. Such device is preferably in a handheld form.

In a further embodiment, the subject invention provides a highlysensitive, specific, and quantitative-capable diagnostic platformutilizing a lateral flow assay with the rare earth nanocrystals bound tooligonucleotides or antibodies capable of being read with, for example,a cell phone camera. The assay does not require DNA amplification andcan be applied to detect a wide range of agricultural pathogens. In aspecific example, the assay can be used to detect Xanthomonas axonopodispv. manihotis (bacterial blight) in cassava.

Detection methods of agricultural diseases historically requirelaboratory analysis, limiting their use in resource-limited settings.Traditional lateral flow assays, while easier to utilize in fieldsettings are typically less sensitive than lab-based methods, such asPCR. When an optical reader is combined with a lateral flow assay aseveral orders of magnitude improvement is achieved over visual reading;however, optical readers are cost prohibitive for distributed use.

In one embodiment, the assay of the subject invention addresses thisproblem by utilizing nanocrystals conjugated to oligonucleotides, whichare then utilized in a lateral flow assay format. The high efficiencyand sensitivity of the nanocrystal eliminates the need for a DNAamplification step and the use of an optical reader. Rather, the readercan utilize non-complex technology such as an LED flash and a camera.The flash and the camera can be, for example, those which are typicallyincorporated into a standard cell phone.

Advantageously, recording the results through a cell phone (or similardevice) facilitates the transfer and aggregation of data. This can beused to create a more balanced dataset, from which, for example, machinelearning can be applied to better predict outbreaks of agriculturaldiseases.

In a specific embodiment, the subject invention, provides a lateral flowassay format where the nanocrystals in the detectable label constitutean up-converting phosphor reporter. The consecutive flow techniqueallows for the use of a reporter such as nanocrystals covered withcapture molecules. In certain embodiments, the flow rate can be fasterand flow time shorter compared to conventional assays.

The solid support provides for the capillary flow of sample out from thesample receiving area to the target capture zones when the lateral flowtest strip is exposed to an appropriate carrier liquid of the sample.

In one embodiment, the lateral flow test strips or microfluidic devicesmay contain one or more sample receiving areas/channels, which allowsthe application of multiple samples. Each of the samples may contain adifferent analyte, or may contain the same analyte. In anotherembodiment, the sample receiving area comprises the absorbent pad thatmay impregnated with buffer salts and surfactants that make the samplesuitable for interaction with the detection system.

In a further embodiment, the lateral flow test strips may contain one ormore target capture zones. The surface of capture zones is modified withan entity that specifically binds to an analyte of interest, forexample, the microbe or microbe-based angents in the environmentalsample. The modification of the surface of capture zones may be achievedby linking the solid support to, for example, antibodies, proteins,nucleotides, and/or other compounds. Such modification may be the sameor different modification applied to nanocrystals. Each of the analytecapture zones may bind a different species of analyte, or may bind thesame species of analyte. In lateral flow test strips where each of theanalyte capture zones binds the same species of analyte, the binding mayoccur at varying concentrations of analyte. The capture zone can be anyshape, as long as it attracts the sample and solvent flow from thesample receiving area through the analyte capture zones.

In one embodiment, the lateral flow test strips exhibit tolerance forvariations in pH (e.g., pH 2-12), ion strength, viscosity, andbiological matrices, contributing to few, if any, false positive andfalse negative results.

Up-conversion luminescence is based on the absorption of two or morelow-energy (longer wavelength, typically infrared) photons by ananocrystal followed by the emission of a single higher-energy (shorterwavelength) photon. Some aspects of lateral flow assays using UCP's havebeen described in Corstjens et al. (2014), Feasibility of Lateral FlowTest for Neurocysticercosis Using Novel Up-Converting Nanomaterials anda Lightweight Strip Analyzer, PloS Negl. Trop. Dis. 8(7):e2944. which isincorporated herein by reference in its entirety.

B. PCR Assays

In another embodiment, the materials and methods of the subjectinvention are combined with PCR procedures to create a highly sensitiveassay. The incorporation of uniform-sized nanocrystal UCPs into PCRproducts generated via amplification using one (or both) PCR primer(s)coupled to the nanocrystals at the 5′ end of the oligonucleotide primersprovides superior assay characteristics when compared to standardreporter molecules used for detection.

Advantageously, unlike commonly used reporter molecules (e.g., alkalinephosphatse and horseraddish peroxidase), the signals produced from thenanocrystals are devoid of background florescence and lack interferencewith other biological molecules. In addition, because the UCP signallasts up to 20 years, the signal can be temporally integrated toincrease the sensitivity of the assay. Advantageously, the uniformity ofthe nanocrystal size and morphology enable stoichiometric coupling ofthe UCP to the oligonucleotide, which improves sensitivity, quantitationand the dynamic range of the assay.

Additionally, nanocrystal reporter pairs with complementary opticalproperties can be utilized in a variety of homogeneous based systems andassays designed to determine co-localization of specific target markerson a single sequence, protein, cell, etc. The complementary nanocrystalpairs exhibit unique optical properties such that, when in proximity toeach other, the emission from nanocrystal A will activate nanocrystal B.In a specific example, a NaYF4:YbTm composition having a 980 nmexcitation and 800 nm emission can excite a NaYF4:YbTmNd compositionhaving an 808 nm excitation and an emission signature around 980 nm.

The optically complementary nanocrystal reporters enable the (1)identification of co-localized targets, (2) identification of specificbinding events in a homogeneous mixture (without separation), and (3)multiplexed identification of the presence of markers along specificoligonucleotide sequences as well as co-localization. For assay targetswhere there is expected to be low target numbers, inexpensiveconcentration of the target species using, for example, well-knownmagnetic bead-based technologies can be readily implemented.

C. Multi-well Assays

In another embodiment, the assay of the invention may be performed onmulti-well arrays, for example, 8, 12, 24, 48, 96, 192, 384-well arrays,in a high-throughput setting.

Analytes

The present invention provides methods and devices to efficiently andaccurately detect, quantify and/or track microbes, microbe-based agents,and/or other analytes in environmental samples.

The analytes can be microbes, microbe-based agents and/or analytesarising from the presence or activity of microbes. The microbes can bebeneficial microorganisms or pathogens, including agriculturalpathogens.

Microbes that can be detected, quantified and/or tracked according tothe subject invention include, but are not limited to bacteria, archaea,yeast, fungi, viruses, protozoa, and multicellular organisms. Themicrobe-based agents that can be analytes according to the subjectioninvention include, but are not limited to, composition containingmicrobes, microbe metabolites and other microbe growth by-products. Inone embodiment, the present invention further provides methods fordetecting a product produced by an entity (such as an animal or plant)in response to a microbe and/or microbe-based agent.

In one embodiment, the present invention further provides methods fordetecting a product produced by an entity (such as an animal or plant)in response to a microbe and/or microbe-based agent.

In one embodiment, the method detects a product, produced by an entityinfected by an agricultural pathogen. The entity can be a plant or apart of the plant including leaf, stem, root, and flower. Theenvironmental sample may include, but is not limited to, soluble plantextracts, and insoluble plant extract.

In certain embodiments, the product produced by an entity in response toa microbe and/or microbe-based agent may be a protein, polypeptide,nucleotide and/or other molecule. The product may be secreted into theenvironment or food sample.

A. Beneficial Microbes

The microbes that can be detected according to the subject inventioninclude, but not limited to bacteria, archaea, yeast, fungi, viruses,protozoa, or multicellular organisms.

In one embodiment, the microorganisms are bacteria, includinggram-positive and gram-negative bacteria. These bacteria may be, but arenot limited to, for example, Escherichia coli, Rhizobium (e.g.,Rhizobium japonicum, Sinorhizobium meliloti, Sinorhizobium fredii,Rhizobium leguminosarum biovar trifolii, and Rhizobium etli),Bradyrhizobium (e.g., Bradyrhizobium japanicum, and B. parasponia),Bacillus (e.g., Bacillus subtilis, Bacillus firmus, Bacilluslaterosporus, Bacillus megaterium, Bacillus amyloliquifaciens),Azobacter (e.g., Azobacter vinelandii, and Azobacter chroococcum),Arhrobacter (e.g. Agrobacterium radiobacter), Pseudomonas (e.g.,Pseudomonas chlororaphis subsp. aureofaciens (Kluyver)), Azospirillium(e.g., Azospirillumbrasiliensis), Azomonas, Derxia, Beijerinckia,Nocardia, Klebsiella, Clavibacter (e.g., C. xyli subsp. xyli and C. xylisubsp. cynodontis), cyanobacteria, Pantoea (e.g., Pantoea agglomerans),Sphingomonas (e.g., Sphingomonas paucimobilis), Streptomyces (e.g.,Streptomyces griseochromogenes, Streptomyces qriseus, Streptomycescacaoi, Streptomyces aureus, and Streptomyces kasugaenis),Streptoverticillium (e.g., Streptoverticillium rimofaciens), Ralslonia(e.g., Ralslonia eulropha), Rhodospirillum (e.g., Rhodospirillumrubrum), Xanthomonas (e.g., Xanthomonas campestris), Erwinia (e.g.,Erwinia carotovora), Clostridium (e.g., Clostridium bravidaciens, andClostridium malacusomae), and combinations thereof.

In certain embodiments, the methods are used to detect and/or trackBacillus subtilis in the environment. In one embodiment, the microbecomprises Bacillus subtilis strains such as, for example, B. subtilisvar. lotuses strains B1 and B2, which are effective producers ofsurfactin.

In one embodiment, the microorganism is a fungus (including yeast),including, but not limited to, for example, Starmerella, Mycorrhiza(e.g., vesicular-arbuscular mycorrhizae (VAM), arbuscular mycorrhizae(AM)), Mortierella, Phycomyces, Blakeslea, Thraustochytrium,Penicillium, Phythium, Entomophthora, Aureobasidium pullulans, Fusariumvenenalum, Aspergillus, Trichoderma (e.g., Trichoderma reesei, T.harzianum, T. viride and T. hamatum), Rhizopus spp, endophytic fungi(e.g., Piriformis indica), Saccharomyces (e.g., Saccharomycescerevisiae, Saccharomyces boulardii sequela and Saccharomyces torula),Debaromyces, Issalchenkia, Kluyveromyces (e.g., Kluyveromyces lactis,Kluyveromyces fragilis), Pichia spp (e.g., Pichia pastoris), killeryeasts, such as Wickerhamomyces (e.g., Wickerhamomyces anomalus) andcombinations thereof.

More specifically, the method can be used to detect one or more viablefungal strains capable of controlling pests, bioremediation, enhancingoil recovery and other useful purposes, e.g., Starmerella bombicola,Candida apicola, Candida batistae, Candida floricola, Candidariodocensis, Candida stellate, Candida kuoi, Candida sp. NRRL Y-27208,Rhodotorula bogoriensis sp., Wickerhamiella domericqiae, as well as anyother sophorolipid-producing strains of the Starmerella clade.

In another embodiment, the microorganism is a yeast. A number of yeastspecies are suitable for production according to the current invention,including, but not limited to, Saccharomyces (e.g. Saccharomycescerevisiae, Saccharomyces boulardii sequela and Saccharomyces torula),Debaromyces, Issakhenkia, Kluyveromyces (e.g. Kluyveromyces lactis,Kluyveromyces fragilis), Pichia spp (e.g. Pichia pastoris), andcombinations thereof.

In certain embodiments, the microbes may be chosen from strains ofkiller yeast. In another embodiment, the microbes are Wickerhamomycesanomalus strains.

Wickerhamomyces anomalus, also known as Pichia anomala and Hansenulaanomala, is frequently associated with food and grain production. It iscapable of growing on a wide range of carbon sources at low pH, underhigh osmotic pressure, and with little or no oxygen, allowing for itssurvival in a wide range of environments.

In specific embodiments, the subject invention provides a method todetect the W. anomalus yeast strain and mutants thereof in theenvrionment. Procedures for making mutants are well known in themicrobiological art. For example, ultraviolet light and nitrosoguanidineare used extensively toward this end. In one embodiment, the microbe isthe Starmerella yeast clade, such as Starmerella bombicola.

In one embodiment, the microorganism is an archaea, or eubacteria,including, but not limited to, Methanobacteria, Methanococci,Methanomicrobia, Methanopyri, Halobacteria, Halococci, Thermococci,Thermoplasmata, Thermoproetei, Psychrobacter, Arthrobacter, Halomonas,Pseudomonas, Hyphomonas, Sphingomonas, Archaeoglobi, Nanohaloarchaea,extremophilic archaea, such as thermophiles, halophiles, acidophiles,and psychrophiles, and combinations thereof.

In one embodiment, the microbe is a virus, including but not limited toadenovirus, cytomegalovirus, viruses of the herpes family, varicellazoster, influenza, rhinovirus, measles, mumps, enteroviruses, and thelike.

In specific embodiments, microbes for the production of SLPs can beCandida sp., Cryptococcus sp., Cyberlindnera samutprakarnensis JP52 (T),Pichia anomala, Rhodotorula sp., or Wickerhamiella sp.

In further specific embodiments, microbes for the production of MELs canbe Pseudozyma sp., Candida sp., Ustilago sp., Schizonella sp., orKurtzmanomyces sp.

Other microbial strains including, for example, other microbial strainscapable of digesting polymers or accumulating significant amounts of,for example, glycolipid-biosurfactants, enzymes, solvents, or otheruseful metabolites can also be used in accordance with the subjectinvention. For example, useful metabolites according to the presentinvention include mannoprotein, beta-glucan and other metabolites thathave bio-emulsifying and surface/interfacial tension-reducingproperties.

B. Pathogens

In one embodiment, the present invention provides methods for detectingpathogens in the environmental samples. The pathogens may include, butnot limited to, a member of one the genera Yersinia, Klebsiella,Providencia, Erwinia, Enterobacter, Salmonella, Serratia, Aerobacter,Escherichia, Pseudomonas, Shigella, Vibrio, Aeromonas, Streptococcus,Staphylococcus, Micrococcus, Moraxella, Bacillus, Clostridium,Corynebacterium, Eberthella, Francisella, Haemophilus, Bacteroides,Listeria, Erysipelothrix, Acinetobacter, Brucella, Pasteurella,Flavobacterium, Fusobacterium, Streptobacillus, Calymmatobacterium,Legionella, Treponema, Borrelia, Leptospira, Actinomyces, Nocardia,Rickettsia, Micrococcus, Mycobacterium, Neisseria, or Campylobacter.

The pathogens may also include, but not limited to a pathogenic virussuch as, a member of the Papilloma viruses, Parvoviruses, Adenoviruses,Herpesviruses, Vaccine virus, Arenaviruses, Coronaviruses, Rhinoviruses,Respiratory syncytial viruses, Influenza viruses, Picornaviruses,Paramyxoviruses, Reoviruses, Retroviruses, Rhabdoviruses, or humanimmunodeficiency virus (HIV).

The pathogens may further include, but not limited to a member of one ofthe genera Taenia, Hymenolepsis, Diphyllobothrium, Echinococcus,Fasciolopsis, Heterophyes, Metagonimus, Clonorchis, Fasciola,Paragonimus, Schistosoma, Enterobius, Trichuris, Ascaris, Ancylostoma,Necator, Wuchereria, Brugi, Loa, Onchocerca, Dracunculus, Naegleria,Acanthamoeba, Plasmodium, Trypanosoma, Leishmania, Toxoplasma,Entamoeba, Giardia, Isospora, Cryptosporidium, Enterocytozoa,Strongyloides, or Trichinella.

According to the subject invention, the pathogens may include, but notlimited to a fungus such as, for example, Ringworm, Histoplasmosis,Blastomycosis, Aspergillosis, Cryptococcosis, Sporotrichosis,Coccidiodomycosis, Paracoccidioidomycosis, Mucomycosis, Candidiasis,Dermatophytosis, Protothecosis, Pityriasis, Mycetoma,Paracoccidiodomycosis, Phaeohphomycosis, Pseudallescheriasis,Trichosporosis, or Pneumocystis.

In one embodiment, the pathogens according to the subject invention mayinclude, but not limited to bovine papular stomatitus virus (BPSV),bovine herpes virus (BVH), bovine viral diarrhea (BVD), foot-and-mouthdisease virus (FMDV), blue tongue virus (BTV), swine vesicular diseasevirus (SVD), porcine respiratory reproductive syndrome virus (PRRS),vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus(VESV).

In specific embodiments, the pathogen according to the subject inventionmay be Neisseria meningitides, Streptococcus agalactiae, Staphylococcusaureus, Porphyromonas gingivalis, Chlamydia pneumoniae, Bacillusanthracis, Streptococcus suis, Echinococcus granulosus, Streptococcussanguinis, and Helicobacter pylori.

In one embodiment, the pathogen according to the subject invention mayproduce toxic molecules that pose threat to human health and cropgrowth. For example, Aspergillus flavus and Aspergillus parasiticusproduce aflatoxin B1 (AFB1), a highly toxic aflatoxin, which cancontaminate grains and other crops such as peanut, corn, rice, andsoybean. Other toxins produced by pathogen include, but are not limitedto, ochratoxin A, botulinum toxin, shiga toxin 1, shiga toxin 2, andstaphylococcal enterotoxin B.

Plants

Plants that can be tested according to methods of the subject inventioninclude: Row Crops (e.g., Corn, Soy, Sorghum, Peanuts, Potatoes, etc.),Field Crops (e.g., Alfalfa, Wheat, Grains, etc.), Tree Crops (e.g.,Walnuts, Almonds, Pecans, Hazelnuts, Pistachios, etc.), Citrus Crops(e.g., orange, lemon, grapefruit, etc.), Fruit Crops (e.g., apples,pears, etc.), Turf Crops, Ornamentals Crops (e.g., Flowers, vines,etc.), Vegetables (e.g., tomatoes, carrots, etc.), Vine Crops (e.g.,Grapes, Strawberries, Blueberries, Blackberries, etc.), Forestry (eg,pine, spruce, eucalyptus, poplar, etc), Managed Pastures (any mix ofplants used to support grazing animals).

Further plants that can benefit from the products and methods of theinvention include all plants that belong to the superfamilyViridiplantae, in particular monocotyledonous and dicotyledonous plantsincluding fodder or forage legumes, ornamental plants, food crops, treesor shrubs selected from Acer spp., Actinidia spp., Abelmoschus spp.,Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp.,Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apiumgraveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avenaspp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasahispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g.Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]),Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa,Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Caryaspp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichoriumendivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp.,Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrumsativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp.,Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpuslongan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g.Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef,Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora,Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica,Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g.Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthusspp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp.,Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp.,Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum,Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzulasylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp.,Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp.,Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp.,Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotianaspp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryzasativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum,Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp.,Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleumpratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp.,Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunusspp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp.,Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubusspp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamumspp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanumintegrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp.,Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao,Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticumspp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum,Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcumor Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vacciniumspp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays,Zizania palustris, Ziziphus spp., amongst others.

Further examples of plants of interest include, but are not limited to,corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava(Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turfgrasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewings fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerate); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovine); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pretense); velvet bentgrass (Agrostis canine); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Plants of interest further include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,millet, etc. Oil-seed plants include cotton, soybean, safflower,sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, oliveetc. Leguminous plants include beans and peas. Beans include guar,locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, limabean, fava bean, lentils, chickpea, etc.

Plant Diseases

Examples of plant diseases that can be detected according to the presentinvention, include the following:

Diseases of wheat: Fusarium head blight (Fusarium graminearum, F.avenacerum, F. culmorum, Microdochium nivale), Typhula snow blight(Typhula sp., Micronectriella nivalis), loose smut (Ustilago tritici, U.nuda), bunt (Tilletia caries), leaf blotch (Mycosphaerella graminicola),and glume blotch (Leptosphaeria nodorum);

Diseases of corn: smut (Ustilago maydis) and brown spot (Cochliobolusheterostrophus); Diseases of citrus: melanose (Diaporthe citri), scab(Elsinoe fawcetti), penicillium rot (Penicillium digitatum, P.italicum), and Citrus Greening (Candidatus Liberibacter spp.);

Diseases of apple: blossom blight (Monilinia mali), powdery mildew(Podosphaera leucotricha), Alternaria leaf spot (Alternaria alternataapple pathotype), scab (Venturia inaequalis), bitter rot (Colletotrichumacutatum), and crown rot (Phytophtora cactorum);

Diseases of pear: scab (Venturia nashicola, V. pirina), black spot(Alternaria alternata Japanese pear pathotype), rust (Gymnosporangiumharaeanum), and phytophthora fruit rot (Phytophtora cactorum);

Diseases of peach: brown rot (Monilinia fructicola), scab (Cladosporiumcarpophilum), and phomopsis rot (Phomopsis sp.);

Diseases of grape: anthracnose (Elsinoe ampelina), ripe rot (Glomerellacingulata), black rot (Guignardia bidwellii), downy mildew (Plasmoparaviticola), and gray mold (Botrytis cinerea); Diseases of Japanesepersimmon: anthracnose (Gloeosporium kaki) and leaf spot (Cercosporakaki, Mycosphaerella nawae);

Diseases of gourd: anthracnose (Colletotrichum lagenarium), Target leafspot (Corynespora cassiicola), gummy stem blight (Mycosphaerellamelonis), Fusarium wilt (Fusarium oxysporum), downy mildew(Pseudoperonospora cubensis), and Phytophthora rot (Phytophthora sp.);Diseases of tomato: early blight (Alternaria solani), leaf mold(Cladosporium fulvum), and late blight (Phytophthora infestans);

Diseases of cruciferous vegetables: Alternaria leaf spot (Alternariajaponica), white spot (Cercosporella brassicae), and downy mildew(Peronospora parasitica);

Diseases of rapeseed: sclerotinia rot (Sclerotinia sclerotiorum) andgray leaf spot (Alternaria brassicae);

Diseases of soybean: purple seed stain (Cercospora kikuchii), sphacelomascad (Elsinoe glycines), pod and stem blight (Diaporthe phaseolorum var.sojae), rust (Phakopsora pachyrhizi), and brown stem rot (Phytophthorasojae);

Diseases of azuki bean: gray mold (Botrytis cinerea) and Sclerotinia rot(Sclerotinia sclerotiorum);

Diseases of kidney bean: gray mold (Botrytis cinerea), sclerotinia seedrot (Sclerotinia sclerotiorum), and kidney bean anthracnose(Colletotrichum lindemthianum);

Diseases of peanut: leaf spot (Cercospora personata), brown leaf spot(Cercospora arachidicola), and southern blight (Sclerotium rolfsii);

Diseases of potato: early blight (Alternaria solani) and late blight(Phytophthora infestans);

Diseases of cotton: Fusarium wilt (Fusarium oxysporum); Diseases oftobacco: brown spot (Alternaria longipes), anthracnose (Colletotrichumtabacum), downy mildew (Peronospora tabacina), and black shank(Phytophthora nicotianae);

Diseases of sugar beat: Cercospora leaf spot (Cercospora beticola), leafblight (Thanatephorus cucumeris), Root rot (Thanatephorus cucumeris),and Aphanomyces root rot (Aphanidermatum cochlioides);

Diseases of rose: black spot (Diplocarpon rosae) and powdery mildew(Sphaerotheca pannosa);

Diseases of chrysanthemum and asteraceous plants: downy mildew (Bremialactucae) and leaf blight (Septoria chrysanthemi-indici);

Diseases of various plants: diseases caused by Pythium spp. (Pythiumaphanidermatum, Pythium debarianum, Pythium graminicola, Pythiumirregulare, Pythium ultimum), gray mold (Botrytis cinerea), Sclerotiniarot (Sclerotinia sclerotiorum), and Damping-off (Rhizoctonia solani)caused by Rhizoctonia spp.;

Disease of Japanise radish: Alternaria leaf spot (Alternariabrassicicola);

Diseases of turfgrass: dollar spot (Sclerotinia homeocarpa), brownpatch, and large patch (Rhizoctonia solani);

Disease of banana: sigatoka (Mycosphaerella fijiensis, Mycosphaerellamusicola, Pseudocercospora musae); and

Seed diseases or diseases in the early stages of the growth of variousplants caused by bacteria of Aspergillus genus, Penicillium genus,Fusarium genus, Tricoderma genus, Thielaviopsis genus, Rhizopus genus,Mucor genus, Phoma genus, and Diplodia genus.

The disease may be root borne, foliar, present in the vascular system ofthe plant or transmitted by insects and include all bacterial, viral,and fungal pathogens of plants.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A method for detecting a target analyte in an environmental or foodsample, comprising the steps of: contacting the sample with a pluralityof nanocrystals, wherein the nanocrystals have been surface modifiedwith an entity that specifically binds to the analyte in the sample,separating the nanocrystals bound to the analyte in the sample fromunbound nanocrystals, and detecting the nanocrystals that bind to theanalyte.
 2. The method according to claim 1, wherein the nanocrystalshave unique and uniform morphology, size, and/or composition, producinga unique optical signature.
 3. The method, according to claim 2, whereinthe unique optical signature is manifested in rise and/or decay times.4. The method according to claim 1, wherein the nanocrystals areup-converting phosphor particles.
 5. The method according to claim 1,wherein the nanocrystals comprise at least one rare earth elementselected from lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium(Ne), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd),terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm),ytterbium (Yb), and lutetium (Lu).
 6. The method according to claim 1,wherein the nanocrystals have a size ranging from 4 nm to 400 nm.
 7. Themethod, according to claim 1, wherein the nanocrystals emit light forgreater than 10⁻⁸ seconds.
 8. The method, according to claim 1, whereinthe nanocrystals can be excited at a wavelength from 900 nm to 1000 nm.9. The method, according to claim 8, wherein the nanocrystals areexcited at a wavelength from 960 nm to 980 nm.
 10. The method, accordingto claim 1, wherein the nanocrystals emit light at a wavelength from 400nm to 12,000 nm.
 11. The method, according to claim 1, wherein thenanocrystals are β-phase particles.
 12. The method, according to claim1, wherein the nanocrystals are combined with a second reporter selectedfrom quantum dots, carbon nanotubes, gold particles, silver particles,and magnetic or dye-doped particles.
 13. The method according to claim1, wherein the entity that specifically binds to the analyte is anantibody, protein, aptamer polypeptide, or polynucleotide.
 14. Themethod, according to claim 1, wherein genomic analysis is used toidentify a specific epitope from genetic sequence information of anunculturable microbe or a mixed population of microorganisms, andwherein a binding agent to the genetically-identified epitope isproduced that specifically binds to the unculturable microbe.
 15. Themethod, according to claim 1, wherein the analyte is a bacterium, yeast,fungus, or virus.
 16. The method, according to claim 1, wherein theanalyte is an agricultural pathogen.
 17. The method, according to claim16, wherein the agricultural pathogen is selected from pathogens thatcause citrus greening disease, potato late blight, grape powdery mildew,red blotch, tobacco mosaic virus, fire blight and/or Pierce's Disease.18. The method according to claim 1, wherein the sample is soil or plantmaterial.
 19. The method according to claim 18, wherein the sample isplant tissue.
 20. The method according to claim 1, wherein the analyteis a microbe-based agent.
 21. The method according to claim 20, whereinthe microbe-based agent is a microbial biosurfactant or a mycotoxin. 22.The method, according to claim 1, wherein the sample is food and theanalyte is a mycotoxin.
 23. The method, according to claim 1, whereinthe sample is a biological sample from an animal.
 24. The method,according to claim 23, wherein the biological sample is a blood, fecal,mucous, saliva, or tissue sample.
 25. The method, according to claim 1,wherein the sample is a water sample.
 26. The method, according to claim25, wherein the water sample is selected from drinking water, groundwater, surface water and wastewater.
 27. The method, according to claim1, wherein the sample is a commercial product that contains microbes.28. The method, according to claim 27, wherein the product is for use inagriculture.
 29. The method, according to claim 27, wherein the productis a food product,
 30. The method, according to claim 29, wherein themicrobes are probiotics.
 31. The method, according to claim 29, whereinthe microbes are pathogenic.
 32. The method according to claim 1,wherein the analyte is a microbe and the detection sensitivity for theanalyte is 10¹ CFU/mL or less.
 33. The method, according to claim 1,wherein the nanocrystals are tuned to avoid background interference fromnaturally occurring chromophores in a sample.
 34. The method, accordingto claim 1, wherein multiple independently-tuned nanocrystals are placedin a multiplexed array on a single support to facilitate analysis ofmultiple analytes from a single sample.
 35. The method, according toclaim 1, wherein 5 or more analytes are analyzed simultaneously
 36. Themethod, according to claim 1, wherein the method is performed within 100yards of where the sample was obtained.
 37. The method, according toclaim 1, wherein the method is performed within 10 minutes of when thesample was obtained.
 38. The method according to claim 1, wherein thedetecting step is performed in a single readout.
 39. (canceled)
 40. Themethod, according to claim 1, wherein the detection, quantificationand/or tracking of the analyte is done by a farmer, regulatory official,compliance official, or distributer.
 41. The method, according to claim1, where the assay is conducted at any point in the supply chain fromimmediately post-production of a commercial product to just prior to useof the product.
 42. The method, according to claim 1, wherein data fromindividual tests are transmitted to a database that can be accessed froma location that is remote from the location where the test wasperformed.
 43. The method, according to claim 42, wherein the data isused to assess performance of beneficial microbes or assess the movementof pathogens.
 44. The method according to claim 1, which is accomplishedusing a lateral flow or microfluidic assay.
 45. The method according toclaim 44, wherein the lateral flow or microfluidic assay is animmunoassay.
 46. The method according to claim 44, wherein the assay isperformed using a portable detection device.
 47. The method, accordingto claim 46, wherein the portable detection device comprises an LED anda camera,
 48. The method, according to claim 47, wherein the portabledetection device is a cell phone.
 49. The method according to claim 44,wherein the assay is carried out utilizing a multiple flow technique.50. The method according to claim 44, wherein a lateral flow test striphas a solid support comprising one or more sample receiving areas andone or more target capture zones.
 51. The method according to claim 50,wherein the solid support is nitrocellulose or engineered microfluidicchannels etched or molded into a plastic or glass substrate.
 52. Themethod according to claim 48, wherein the target capture zone has beensurface modified to specifically bind microbes or microbe-based agentsin the environmental sample.
 53. A device for performing the assay ofclaim
 1. 54. The device, according to claim 53, comprising nanocrystalsthat have been surface modified with an entity that specifically bindsto the target analyte.
 55. The device, according to claim 54, whereinthe nanocrystals have unique and uniform morphology, size, and/orcomposition, producing a unique optical signature.
 56. The device,according to claims 55, wherein the unique optical signature ismanifested in rise and/or decay times.
 57. The device, according toclaim 54, wherein the nanocrystals are up-converting phosphor particles.58. The device, according to claim 54, wherein the nanocrystals compriseat least one rare earth element selected from lanthanum (La), cerium(Ce), praseodymium (Pr), neodymium (Ne), promethium (Pm), samarium (Sm),europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium(Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu). 59.The device, according to claim 54, wherein the nanocrystals have a sizeranging from 4 nm to 400 nm.
 60. The device, according to claim 54,wherein the nanocrystals emit light for greater than 10⁻⁸ seconds. 61.The device, according to claim 54, wherein the nanocrystals can beexcited at a wavelength from 900 nm to 1000 nm.
 62. The device,according to claim 61, wherein the nanocrystals are excited at awavelength from 960 nm to 980 nm.
 63. The device, according to claim 54,wherein the nanocrystals emit light at a wavelength from 400 nm to12,000 nm.
 64. The device, according to claim 54, wherein thenanocrystals are β-phase particles.
 65. The device, according to claim54, wherein the nanocrystals are combined with a second reporterselected from quantum dots, carbon nanotubes, gold particles, silverparticles, and magnetic or dye-doped particles.
 66. The device,according to claim 54, wherein the entity that specifically binds to theanalyte is an antibody, protein, aptamer polypeptide, or polynucleotide.67. The device, according to claim 54, wherein the nanocrystals aretuned to avoid background interference from naturally occurringchromophores in a sample.
 68. The device, according to claim 54, whereinmultiple independently-tuned nanocrystals are placed in a multiplexedarray on a single support to facilitate analysis of multiple analytesfrom a single sample.
 69. The device, according to claim 54, whereinsaid device can transmit data from individual tests to a database thatcan be accessed from a location that is remote from the location wherethe test was performed.
 70. The device, according to claim 54, which isa lateral flow or microfluidic assay.
 71. The device, according to claim70, wherein the lateral flow or microfluidic assay is an immunoassay.72. The device, according to claim 54, comprising, as one component ofthe device, a portable detection unit.
 73. The device, according toclaim 72, wherein the portable detection unit comprises an LED and acamera.
 74. The device, according to claim 73, wherein the portabledetection unit is a cell phone.
 75. The device, according to claim 54,wherein the assay is carried out utilizing a multiple flow technique.76. The device, according to claim 54, wherein a lateral flow test striphas a solid support comprising one or more sample receiving areas andone or more target capture zones.
 77. The device, according to claim 76,wherein the solid support is nitrocellulose or engineered microfluidicchannels etched or molded into a plastic or glass substrate.
 78. Thedevice, according to claim 76, wherein the target capture zone has beensurface modified to specifically bind microbes or microbe-based agentsin the environmental sample.
 79. An assay for detecting a targetpolynucleotide sequence using PCR, wherein said method comprises the useof primer sequences to amplify said target polynucleotide sequencewherein at least one of said primer sequences is coupled to ananocrystal.